Immune-dependent responses are selective in defining non-self or aberrant antigen presentation. Unfortunately, long-term antibody production to human cancer antigens is limited. In an innovative and novel approach, we have developed a method to utilize primary immune reactions in tumor draining lymph nodes to provide the means to define biologically active tumor antigens originating from breast cancers. This novel approach combines a series of steps to coordinate the construction of low complexity antibody cDNA libraries and protein production that are used to identify tumor antigens using sensitive antibody microscale "antigen-trap" assays followed by LC-MS/MS antigen identification. Tumor antigens identified can then be verified as potential tumor antigens using biochemical, immunological and molecular methodologies. The methodologies applied are medium throughput platform based designed to rapidly evaluate matched lymph node and tumor samples from the same patient. This project applies innovative technologies that demonstrate that: a) tumor draining lymph nodes are immuno-reactive to aberrant breast cancer antigens and produce antigen-dependent somatic hypermutation in proliferative B-cell germinal centers, b) antigen binding domains of somatic hypermutated antibodies synthesized as recombinant VH and/or VHVl/Vk ScFv proteins can specifically recognize and identify breast cancer antigens, and c) antigens identified can be verified as diagnostic for breast cancer sub-phenotypes. This R21 application proposes to expand and refine our methodologies to: 1) determine the diversity and effectiveness of immune-selection of tumor antigens in a larger patient population, 2) expand our ability to produce antibody molecules with appropriate structure and antigen binding, and 3) develop methodologies to incorporate antibody proteins synthesized into highly sensitive assays that can screen primary cancer, histological material and/or biological fluids necessary to evaluate the potential of antigens as diagnostic biomarkers.